Development and applications of an optimal Hypocrea jecorina-based enzyme expression system

Improving efficiency in enzymatic hydrolysis of biomass remains paramount to producing economically viable cellulosic biofuels, yet the full mechanistic understanding and fundamental knowledge of fungal cellulases remains incomplete. Given the vast differences in enzymatic activity of the same enzymes produced in varied heterologous hosts, it is imperative to study these enzymes in their native and industrially relevant host strains. However, this remains technically challenging in commonly used expression strains.  Previously, we have developed a robust expression system using the filamentous fungal host Hypocrea jecorina (Trichoderma reesei), capable of high levels of single-enzyme production of cellulolytic enzymes indistinguishable from those expressed in native strains.  In this study, we expand on our previous results and detail the latest improvements to our H. jecorina-based enzyme expression system. Here we detail the expression and characterization of heterologous and mutant enzymes, genetic improvements to our host strains, and the development of new tools to expand our abilities to engineer H. jecorina. In essence, we detail the progressive history of the development of our current expression system including pitfalls and breakthroughs, our current state of technology and where we envision taking this system in the future.